The Ca2+ Synapse Redo
A Matter of Location, Location, Location
The past decade has witnessed the evolution of a new paradigm in thinking about the intimate interrelationship between cellular structure and physiological function in biological processes. It is not surprising that this evolution has, perhaps, its clearest history in the field of excitation-contraction (E-C) coupling, which has had to wrestle with the long-standing paradox of how the release of Ca2+ from the sarcoplasmic reticulum (SR) can be graded by membrane potential1 in the presence of regenerative Ca2+ release (Ca2+-induced Ca2+ release2–4⇓⇓). In retrospect, it is clear that the resolution of this paradox had to await both methodological and theoretical breakthroughs that allowed researchers to change the scale of our measurements and thinking. Until the early 1990s, measurements of intracellular Ca2+ were limited to spatially averaged whole-cell Ca2+ transients or Ca2+ waves. The corresponding theoretical constructs assumed a spatially continuous distribution of SR Ca2+ release, which successfully predicted the properties of Ca2+ waves but precluded graded Ca2+ transients—hence the paradox.
In 1992, Stern5 made a key advance toward the resolution of this paradox by recognizing that all-or-nothing SR Ca2+ release could be avoided by having discrete Ca2+ release sites that were spatially segregated. This model was unique in that it included the intimate association or coupling of dihydropyridine receptors (DHPRs) to a discrete cluster of ryanodine receptors (RyRs), forming what he called a “ Ca2+ synapse.” In this model, each cluster of RyRs may release Ca2+ in an all-or-nothing manner, but because of their physical separation, each cluster could act as an independent unit. Gradation of the magnitude of the whole-cell Ca2+ transient is, then, determined by the number of activated Ca2+ synapses, just as skeletal muscle contraction is graded by the number of firing motor neuron synapses. Stern’s important contribution was the recognition of the functional consequences of the cellular molecular architecture.
Methodological advances in laser scanning confocal microscopy and the development of a fluorescent Ca2+ indicator with high quantum yield (fluo-3) provided new tools for measuring cytosolic Ca2+ at an unprecedented submicron scale. The observation of Ca2+ sparks by Cheng et al6 and others7–10⇓⇓⇓ and the localization of Ca2+ sparks to t-tubules11 provided the experimental underpinnings for Stern’s concept of the Ca2+ synapse.5 These observations set the stage for the “local control” theory of E-C coupling, where SR Ca2+ release is controlled by the L-type Ca2+ current because independent, elementary events of SR Ca2+ release are “recruited” by Ca2+ flowing through single L-type Ca2+ channels, and not by the average intracellular Ca2+ concentration.12 The molecular underpinnings for the local control theory were demonstrated by the colocalization of DHPRs and RyRs in several muscle types.13,14⇓ Clearly, this past work has been focused primarily on the role of molecular architecture as it pertains to the local control of SR Ca2+ release. Recent work, as with the study by Yang et al15 in this issue of Circulation Research, has expanded the notion of local control to include some of the cellular processes that are important for the regulation of Ca2+ removal and/or uptake such as the sodium-calcium exchanger (NCX).
The sodium-calcium exchanger is a major route for Ca2+ removal from the cytoplasm in cardiac muscle and might also contribute to the Ca2+ trigger for SR Ca2+ release. Accordingly, NCX would be predicted to be distributed in the vicinity of the Ca2+ synapse machinery of DHPRs and RyRs at the t-tubule–junctional SR region. However, attempts at the molecular localization of the NCX have yielded disparate results with some studies showing NCX distributed primarily in the t-tubules16,17⇓ and other studies showing NCX distributed throughout all membranes in contact with the extracellular space.18 Yang et al15 provide functional information regarding the localization of NCX and Na+ channels that goes a long way in resolving the controversies of the spatial localization of NCX. They exploited the ability to functionally inactivate t-tubules via osmotic shock (detubulate) in rat ventricular cells and they showed that in detubulated cells (1) the spatial pattern of SR Ca2+ release resembled that observed in cardiac atrial cells that lack t-tubules, (2) the rate of decay of the whole-cell Ca2+ transient (visualized as fluo-3 fluorescence) was markedly slowed compared with cells with t-tubules, (3) the magnitude of the Na+ current declined in proportion to the decrease in membrane capacitance, and most interestingly (4) the NCX current was virtually abolished. These results emphasize the importance of the NCX in the t-tubules for Ca2+ removal during E-C coupling and leave open the possibility that the NCX on the surface membrane may have an entirely different function. The functional results of Yang et al15 complement nicely the immunocytochemistry experiments of Moore and coworkers, 17 which showed the localization of Na+ channels and NCX in the t-tubules of rat ventricular cells. Importantly, however, the NCX and the Na+ channels were not found in the immediate vicinity of the Ca2+ synapse, but they were localized in the t-tubules.17
The functional localization of NCX by Yang et al15 combined with the molecular localization studies of Scriven et al17 now enable us to expand the domain of Stern’s Ca2+ synapse5 to include the cellular processes that determine both SR Ca2+ release and cytoplasmic Ca2+ removal/uptake. Shown schematically in the Figure, the expanded version of the Ca2+ synapse depicts SR Ca2+ release via the intimate association of DHPRs and RyRs, which are in close proximity with one of the major processes influencing Ca2+ removal, namely NCX and also with Na+ channels. This model is also consistent with the putative role of the NCX to contribute, in part, to the Ca2+ trigger for SR Ca2+ release. As new information regarding the functional and molecular localization of additional ion channels, transporters, and regulatory molecules becomes available, this somewhat simple scheme will become undoubtedly richly populated with the full array of processes that are devoted to the singular task of transiently elevating Ca2+ for muscle contraction.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- ↵Ford LE, Podolsky RJ. Regenerative calcium release within muscle cells. Science. 1970; 167: 58–59.
- ↵Fabiato A. Calcium-induced release of calcium from the sarcoplasmic reticulum. Am J Physiol. 1985; 245: C1–C4.
- ↵Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993; 262: 740–744.
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- ↵López-López JR, Shacklock PS, Balke CW, Wier WG. Local calcium transients triggered by single L-type calcium channel currents in cardiac cells. Science. 1995; 268: 1042–1045.
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- ↵Yang Z, Pascarel C, Steele DS, Komukai K, Brette F, Orchard CH. Na+-Ca2+ exchange activity is localized in the t-tubules of rat ventricular myocytes. Circ Res. 2002; 91: 315–322.
- ↵Frank JS, Mottino G, Reid D, Molday RS, Philipson KD. Distribution of the Na+-Ca2+ exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal gold-labeling study. J Cell Biol. 1992; 117: 337–345.